Characterization or determination of the amount of blood cells by means of poultry antibodies

ABSTRACT

A process for detection of blood cells in activated form and/or determination of a proportion of activated blood cells which comprises performing said detection and/or determination with bird antibodies, whereby interfering interactions which change expression of antigen on a surface of blood cells are eliminated.

TECHNICAL FIELD

The present invention relates to a process for the characterization ordetermination of the amount of mammalian cells according to thetechnique previously known per se where antibodies are utilized for saidpurpose. Up to now antibodies prepared in a mammal, especially rabbit,have been utilized for such purposes, but the present invention relatesto the utilization of other types of antibodies in these connections,whereby disadvantages in connection with the prior art are eliminated,even in such a way that the invention enables such characterizations ordeterminations which would otherwise be troublesome or even impossible.

BACKGROUND OF THE INVENTION

Although the invention is generally applicable to the characterizationor determination of many different types of mammalian cells, which willbe more specifically exemplified below, it is of a very great value inconnection with thrombocytes (blood platelets), especially withreference to the central parts of the thrombocyte functions in differentconnections. Therefore, a background of the invention will be given withreference to thrombocytes, as the problems to be solved by the inventionand the advantages thereof will be most obvious thereby. However,corresponding or similar advantages can be achieved in the determinationof other mammalian cells.

Thrombocytes have a central part of the haemostasis. It is well knownthat low thrombocyte numbers in the blood give rise to bleedingcomplications. There are also data which indicate that an increasednumber of activated thrombocytes and/or a reduced activation thresholdmay have an importance for the development of thrombi. Also suchdiseases as preeclampsia (a syndrome of pregnants with the symptoms ofhigh blood pressure and urine protein) are associated with suchalterations of the normal thrombocyte functions. Examinations of thethrombocyte functions can also be expected to become of great importancefor decisions as to which patients should receive thrombocyteconcentrates.

Haemostasis requires aggregation of thrombocytes. However, saidaggregation can take place only if the thrombocytes will exposereceptors for fibrinogen on their surfaces. In their condition of restthe thrombocytes have a minor capability of binding fibrinogen and thenthey can not either form aggregates which are the primary haemostaticplug (the first blocking of a vascular lesion) or part of a thrombosis(a clot). The activation of thrombocytes means that the thrombocyteswill get a significantly increased capability of binding fibrinogen.

In other words there is a great demand of determining, in the blood of apatient, the amount or portion of the thrombocytes which are activated,i.e. which are highly capable of binding fibrinogen. However, thoseroutine methods which have been utilized for testing the thrombocytefunction, e.g. bleeding time and thrombocyte aggregation, are complexand insensitive and merely give a non-specific information about thefunction. For instance, they will give no picture of the discrete orseparate thrombocytes but merely an average value. Furthermore, thereare methods for determining proteins which are liberated from thethrombocytes, but the results thereof are unreliable as they areinfluenced inter alia by the metabolism of the patient.

In recent years fluorescence activated flow cytometry has, however, madeit possible to develop new assays of thrombocyte functions. In thiscontext laser beams are utilized which illuminate cells in a passingliquid jet. The light scattering gives information about the sizes ofthe cells and their structures. Further information will be obtained bythe addition of antibodies or other substances which have been linked todifferent substances which when illuminated are caused to produce light(fluorescence). These methods are rapid and sensitive and can give aspecific information about thrombocyte functions. Moreover, it ispossible to study each thrombocyte separately and not only an average ofall thrombocytes. For an assay a few microliters of whole blood orthrombocyte-rich plasma will be required. The thrombocytes can bedifferentiated from other blood cells by means of a specific antibody orby their sizes.

Commercially available fluorescence-labelled rabbit antibodies have beenutilized for the study of the fibrinogen binding of thrombocytes inthese connections. This is disclosed in an article by R. M. Hardisty etal. in "Measurement of fibrinogen binding to platelets in whole blood byflow cytometry: a micro method for the detection of plateletactivation." Br. J. Hematol 1990, 76, 387-394.

GENERAL DISCLOSURE OF THE INVENTION

According to the present invention it has unexpectedly been shown thatby the choice of another kind or type of antibodies than those mammalianantibodies which have previously been utilized for the characterizationor determination of mammalian cells, essential advantages are achievedrelative to the prior art.

More specifically it has been found that by selecting antibodies frombirds or reptiles one can avoid or eliminate method artefact appearingin connection with mammalian antibodies. Thus, mammalian antibodies formimmuno complexes with fibrinogen in plasma, which activate thethrombocytes and give rise to erroneous assay results. Apparently thisis not all or to a very reduced extent the case when utilizing the novelantibodies according to the present invention. Although the invention isnot limited to any specific theory in this context, it seems as if theantibodies from birds and reptiles do not provoke physiologicalreactions in the formation of complexes with antigen and interactionswith antibody-specific cell receptors.

In other words the present invention is based on the unexpecteddiscovery that in experiments with antibodies against fibrinogen, whichantibodies had been prepared by the immunization of hens with humanfibrinogen, fibrinogen could be found on the thrombocyte surfaceswithout any activations of the thrombocytes, and this in spite of thefact that immuno complexes could be observed between plasma fibrinogenand fluorescence-labelled hen antifibrinogen antibody (in theexperiments thrombocytes and immuno complexes could be distinguishedfrom each other due to different light scatterings or by means of athrombocyte antibody labelled in any other way, e.g. with another dye orwith a radionucleide).

The use of hen or chicken antibodies in an assay of the ELISA type ispreviously known per se (see e.g. Comp. Immun. Microbiol. Infect. Dis.,Vol. 13, No. 4, pp 199-201, 1990) but what is unexpected in connectionwith the invention is, thus, that a troublesome influence upon theexamined or assayed cells is avoided.

This means that the process according to the invention is intended forthe characterization or determination of such cells which interact withantibodies from mammals, or with their complexes with antigen, in anyother way than by means of the antigen-binding capability of theantibodies. This generally means that such cells are referred to whichare able to undergo a transformation or conversion, i.e. a change of thesurface structure, and where antibodies from mammals, or their complexeswith antigens, induce such a transformation or conversion.

The purpose of the process according to the invention is to assay ordetect in what condition or state the cells are present or to assay ordetermine the amount or proportion of cells in at least one of saidstates, method artefacts or conversions of said cells being avoided bythe invention, which would otherwise give an overrated or erroneousvalue of the proportion of cells in either of said states or conditions.

Although the invention has been generally described above in connectionwith chicken or hen antibodies, it should be useful for antibodies frombirds or poultry in general as well as for antibodies from reptiles.Furthermore, it is not necessary to utilize the antibodies per se, ascorresponding or similar results would be obtained by immunologicallysimilar proteins, i.e. such proteins which possess essentially the sameor similar antigen-binding activity as the antibodies referred to. Forinstance it may be fragments of antibodies and recombinant antibodies.

More specifically this means that the process according to the inventionis characterized by utilizing, as the antibodies, antibodies from birdsor reptiles or immunologically similar proteins having essentially thesame or similar antigen-binding activity or capability as saidantibodies and by performing the characterization and/or determinationon such mammalian cells which interact with antibodies from mammals, orwith their complexes with antigens in any other way than by means of theantigen-binding activities of the antibodies, said characterizationmeaning that one detects the state or condition in which the cells arepresent and/or the determination meaning that one determines the numberor proportion of the cells in at least one of said states or conditions.

As concerns the expression "characterization" of mammalian cells itshould be interpreted in a wide sense, especially as thecharacterization per se can be performed in accordance with knownprinciples, e.g. to detect certain surface structures of the cells.Thus, the invention is applicable to any characterization where themammalian cells interact in the way described or are present in aconvertible form and where the convertion could influence upon themeasured values or results.

According to a preferable embodiment of the invention there are used assaid bird antibodies antibodies from hens or chickens, such as fromchicken blood.

Furthermore, it can often be advantageous rather to utilize hen orchicken antibodies from eggs, as the amount of antibodies therebyobtainable is considerably larger than if chicken blood were used as thesource.

Especially with reference to what has been mentioned above in connectionwith thrombocytes it may also be preferable to utilize chickenantifibrinogen antibodies (throughout the specification and claims theterm chicken is used to include also hen).

In connection with all preferable embodiments concerning chickenantibodies it should be understood that said applications also compriseuses of the corresponding immunologically similar proteins.

The process according to the invention is applicable to thecharacterization or determination of the amount or number of anymammalian cells, e.g. blood cells, such as thrombocytes and white bloodcells, or leucocytes, or endothelial cells, etc., but for theabove-mentioned reasons the characterization and/or determination ofthrombocytes is of course especially interesting, the purpose generallybeing to detect thrombocytes in activated forms or to determine theproportion of activated thrombocytes. In this context activationprimarily means the capability of binding fibrinogen.

Furthermore, it can be added that the process according to the inventionis preferably performed in the form of fluorescence activated flowcytometry (FACS=Fluorescent Antibody Cell Sorter), especially withreference to the fact that said method is rapid, sensible and givesspecific information.

EXAMPLES

For the purpose of comparing the invention with the use of antibodiesaccording to prior art thrombocytes in plasma were admixed withantibodies from chicken and rabbit, respectively, directed against thehuman plasma proteins fibrinogen, α-2-macroglobulin and IgA. Theconcentrations of said antibodies were selected in such a way thatimmuno complexes would be formed to a great extent. The degree ofactivation was measured by means of fluorescence-labelled chickenantibodies directed against fibrinogen. As a negative control there wasadded merely a saline and as a positive control the knownthrombocyte-activating substance adenosine diphosphate.

A detailed method description will follow below and the results arepresented in the accompanying figure, from which it can be gathered thata strong activation was obtained with all immuno complexes containingrabbit antibodies. On the contrary, the only combination with chickenantibodies for which an activation was obtained was in the complex withIgA, but in this specific case the human immunoglobulin IgA in itselfshould be responsible for said activation.

DETAILED METHOD DESCRIPTION

Reagents

Normal mouse IgG, adenosine-5-diphosphate (grade 1) and fluoresceinisothiocyanate (FITC) were purchased from Sigma (St. Louis, Mo., USA).Sephedex G-25 and DEAE-Sepharose were purchased from Pharmacia (Uppsala,Sweden). Chicken antibodies, IgG-fractions purified from egg yolks,directed against human fibrinogen, human IgA, human alfa-2-macroglobulinand mouse immunoglobulin were obtained from Immunsystem AB (Uppsala,Sweden). The antifibrinogen antibodies were affinity purified oninsolubilized human fibrinogen (Larson, A. et al J Immunol Meth 1988,113:93-99). Some of these were conjugated with FITC after having beendisplayed during a night against 0.1 mole/L of NaHCO₃, pH 9.5. Theconjugation ration was 11.1, i.e. 11 parts by mole of FITC per 1 part bymole of antibody. The mixture was incubated at room temperature indarkness for three hours. Conjugated antibodies were separated from freeFITC by gel filtration in 0.01 mole/L of Tris, pH 7.3, by gel filtrationof Sephadex G-25. Antibodies having the proper F/P-ratio were separatedby means of ion exchange chromatography on DEAE-Sepharose CL 6B. Thecolumn was washed with the Tris-buffer with an addition of 0.1 mole/L ofNaCl and the antibodies were eluted by means of a saline gradient to0.25 mole/L. Non-conjugated and FITC-conjugated rabbit antibodiesagainst human fibrinogen, human IgA, human alpha-2-macroglobulin andmouse immunoglobulin were purchased from Dakopetts AS (Glostrup,Denmark).

Blood testing

Venous blood was taken with an open needle without stasis from healthyvolunteers who had not taken any medicine during the last 2 weeks.

4.5 mL of blood were collected in plastic tubes containing 0.5 mL of3.8% sodium citrate. Thrombocyte-rich plasma was prepared bycentrifugation for 10 minutes at 140 x g.

Preparation of Samples for Flow Cytometry

5 μL of thrombocyte-rich plasma were added to plastic tubes containing50 or 55 μL of HEPES-buffer (137 mmole/L of NaCl, 2.7 mmole/L of KCl, 1mmole/L of MgCl₂, 5.6 mmole/L of glucose, 1 mg/mL of bovine serumalbumine and 20 mmole/L of HEPES, pH 7.40) and for some experiments 5 μLof antibodies. In some cases ADP had been added to the HEPES-buffer. Thesamples were carefully mixed once and were left to stand at roomtemperature for exactly 10 minutes. Then 10 μL of FITC-conjugatedchicken antifibrinogen antibody (0.35 g/L) were added and the test tubeswere left to stand for additionally exactly 20 minutes. The reaction wasterminated by 500 μL of icecold PBS with or without 1% ofparaformaldehyde. No washing steps were needed. The samples were kept indarkness on ice until they were analyzed in the flow cytometer, whichwas always performed during the same day.

Flow cytometry

The fluorescence-labelled thrombocytes in the buffer were assayed bymeans of a FACScan cytometer (Becton Dickinson, Mt. View, Calif., USA)equipped with a 15 mW air cooled 488 nm argon laser with lightscattering forwardly (FwSc) and laterally (SSc), and green (FITC)- andred (PHYCO)-signals were collected with a logarithmic amplification withone 530/30 nm and one 585/42 nm filter for a green and red signal,respectively. Collections from 10,000 cells per sample and computerprocessings thereof were made with a Consort 30 program (also BectonDickinson) on a Hewlett Packard 310 PC. Based on the light scatteringproperties each cell was represented by a dot in a rectangular system ofcoordinates. A collection window is placed around the crowd of dotsrepresenting the thrombocytes. Controls have been made withfluorescence-labelled antibodies against the thrombocyte receptorsGPIIIa and GPIb. The instrument gives the percentage of positive cells(for fibrinogen generally thrombocytes in a buffer with the addition ofadenosine and theophyllamine or EDTA have been chosen as the negativecontrol, for the above-mentioned glycoproteins a fluorescence-labelledantibody against the leucocyte surface antigen CD-3), the averagefluorescence intensity, the complexity (SSc) and the average particlevolume (FwSc) of the cell population within the analysis window.

Other Experimental Results

The activation of immuno complexes can at least partly be counteractedif the thrombocytes are firstly mixed with a monoclonal antibody againstthe Fc-receptor. Immuno complexes between mouse antibodies and rabbitantibodies activate thrombocytes but not complexes between mouseantibodies and chicken anti-mouse-antibodies. At least some monoclonalantibodies against thrombocyte surface antigen may give some activationwithout the addition of any other antibody. Said activation is stronglyenhanced by rabbit-anti-mouse-antibodies but notchicken-anti-mouse-antibodies.

We claim:
 1. A process for detection of blood cells in activated formand/or determination of a proportion of activated blood cells whichcomprises performing said detection and/or determination with poultryantibodies, whereby intefering interactions which change expression ofantigen on a surface of blood cells are eliminated.
 2. A processaccording to claim 1 wherein said poultry antibodies are chickenantibodies.
 3. A process according to claim 2 wherein said chickenantibodies are from eggs.
 4. A process according to claim 3 wherein saidblood cells are thrombocytes or leucocytes.
 5. A process according toclaim 4 wherein the poultry antibodies are chicken antifibrinogenantibodies.
 6. A process according to claim 2 wherein said detectionand/or determination is performed in the form of fluorescence-activatedflow cytometry.
 7. A process according to claim 2 wherein said bloodcells are thrombocytes or leucocytes.
 8. A process according to claim 3wherein said detection and/or determination is performed in the form offluorescence-activated flow cytometry.
 9. A process according to claim 3wherein the poultry antibodies are chicken antifibrinogen antibodies.10. A process according to claim 1 wherein said blood cells arethrombocytes or leucocytes.
 11. A process according to claim 10 whereinsaid blood cells are thrombocytes.
 12. A process according to claim 11wherein said detection and/or determination is performed in the form offluorescence-activated flow cytometry.
 13. A process according to claim11 wherein the poultry antibodies are chicken antifibrinogen antibodies.14. A process according to claim 10 wherein said detection and/ordetermination is performed in the form of fluorescence-activated flowcytometry.
 15. A process according to claim 10 wherein the poultryantibodies are chicken antifibrinogen antibodies.
 16. A processaccording to claim 1 wherein said detection and/or determination isperformed in the form of fluorescence-activated flow cytometry.
 17. Aprocess according to claim 1 wherein the poultry antibodies are chickenantifibrinogen antibodies.